Collagenase II

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition
Issued and maintained by the United States Pharmacopeial Convention (USP)
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VQNESKRYTV SYLKTLNYYD LVDLLVKTEI ENLPDLFQYS SDAKEFYGNK
TRMSFIMDEI GRRAPQYTEI DHKGIPTLVE VVRAGFYLGF HNKELNEINK
RSFKERVIPS ILAIQKNPNF KLGTEVQDKI VSATGLLAGN ETAPPEVVNN
FTPILQDCIK NIDRYALDOL KSKALFNVLA APTYDITEYL RATKEKPENT
PWYGKIDGFI NELKKLALYG KINDNNSWII DNGIYHIAPL GKLHSNNKIG
IETLTEVMKV YPYLSMQHLQ SADQIKRHYD SKDAEGNKIP LDKFKKEGKE
KYCPKTYTFD DGKVIIKAGA RVEEEKVKRL YWASKEVNSQ FFRVYGIDKP
LEEGNPODIL TMVIYNSPEE YKLNSVLYGY DTNNGGMYIE PEGTFFTYER
EAQESTYTLE ELFRHEYTHY LQGRYAVPGQ WGRTKLYDND RLTWYEEGGA
ELFAGSTRTS GILPRKSIVS NIHNTTRNNR YKLSDTVHSK YGASFEFYNY
ACMFMDYMYN KDMGILNKLN DLAKNNDVDG YDNYIRDLSS NYALNDKYQD
HMQERIDNYE NLTVPFVADD YLVRHAYKNP NEIYSEISEV AKLKDAKSEV
KKSQYFSTFT LRGSYTGGAS KGKLEDQKAM NKFIDDSLKK LDTYSWSGYK
TLTAYFTNYK VDSSNRVTYD VVFHGYLPNE GOSKNSLPYG KINGTYKGTE
KEKIKFSSEG SFDPDGKIVS YEWDFGDGNK SNEENPEHSY DKVGTYTVKL
KVTDDKGESS VSTTTAEIKD LSENKLPVIY MHVPKSGALN QKVVFYGKGT
YDPDGSIAGY QWDFGDGSDF SSEQNPSHVY TKKGEYTVTL RVMDSSGQMS
EKTMKIKITO PVYPIGTEKE PNNSKETASG PIVPGIPVSG TIENTSDQDY
FYFOVITPGE VKIDINKLGY GGATWVVYDE NNNAVSYATD DGQNLSGKFK
ADKPGRYYIH LYMFNGSYMP YRINIEGSVG R

C₅₀₂₉H₇₆₆₆N₁₃₀₀O₁₅₆₄S₂₁    112,023 (for ζ subtype) CAS RN: 9001-12-1.

1 DEFINITION

Collagenase II (EC 3.4.24.3), isolated from Clostridium histolyticum and encoded by the colH gene (GenBank accession number BAA06251.1), is a key raw material used in the dissociation or destruction of a broad range of tissue types. Collagenase II is a metalloprotease that acts as an endoprotease and also exhibits a tripeptidylcarboxypeptidase activity. It shows endopeptidic activity with the main cleavage site found in front of the human Collagen duplex amino acids glycine-proline. Hydrolysis takes place in the interior of the triple helical domains of collagen.

Collagenase II is also known as class II collagenase and consists of three subtypes: δ, ε, and ζ. Collagenase II ( is the full-length enzyme, whereas collagenase II 8 (100,000 Da) and collagenase II ε (110,000 Da) are thought to be proteolytic degradation products of collagenase II ζ caused by other proteases present in C. histolyticum (mainly a trypsin-like enzyme and clostripain/endoproteinase Arg C).

Collagenase II can be provided in a liquid formulation consisting of 5 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) and 1 mM calcium chloride pH 7.5 and stored as a frozen liquid. The specific activity of collagenase II is 10-16 units/mg of protein using 4-phenylazobenzyloxycarbonyl (PZ)-Pro-Leu-Gly-Pro-D-Arg as the substrate described in the Assay. The peak area for collagenase II is NLT 90% as determined by HPLC described in the test for Purity. The test for Clostripain Activity is used to assess the activity of the clostripain impurity and the acceptance criterion is NMT 0.5 units/mg of protein. The test for Trypsin Activity is used to assess the activity of the trypsin-like enzyme impurity and the acceptance criterion is NMT 0.5 units/mg of protein.

2 IDENTIFICATION

A. It meets the requirements in the Assay.

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the test for Purity.

3 ASSAY

Change to read:

3.1 PROCEDURE

Tris buffer: 0.1 M Tris pH 7.1, prepared as follows. Dissolve 6.05 g of tris(hydroxymethyl)aminomethane (Tris) in 400 mL of water, and adjust with 2 N hydrochloric acid to a pH of 7.1 (at 25 ± 1°). Dilute with water to a final volume of 500 mL.

Substrate solution: Dissolve 10 mg of PZ-Pro-Leu-Gly-Pro-D-Arg in 0.2 mL of methanol, and dilute this solution with Tris buffer to a final volume of 10 mL. [NOTE-Use a freshly prepared solution only.]

Calcium chloride solution: 0.1 M, prepared as follows. Weigh 1.47 g of calcium chloride dihydrate in a volumetric flask, and dilute with water to a final volume of 100 mL.

Citric acid solution: 0.025 M, prepared as follows. Weigh 525 mg of citric acid monohydrate in a volumetric flask, and dilute with water to a final volume of 100 mL.

Extraction mixture: To one test tube per sample to be assayed, pipette 5.0 mL of ethyl acetate and 1.0 mL of Citric acid solution. [NOTE-Use a freshly prepared mixture only.]

Drying tube: Into one test tube per sample to be assayed, add 0.35-0.40 g of sodium sulfate anhydrous. Seal the test tube with parafilm.

Standard solution: Dilute USP Collagenase II RS with Tris buffer to an appropriate dilution to achieve the absorbance range of 0.3-0.9 from the Analysis. (IRA 1-Sep-2022) [NOTE-Avoid freezing and thawing USP Collagenase II RS. After withdrawing USP Collagenase II RS. wipe off the outside of the plastic pipette tip to remove any residual solution.]

Sample solutions: Dilute Collagenase II with Tris buffer to an appropriate dilution to achieve the absorbance range of 0.3-0.9 from the Analysis. Prepare in triplicate. [NOTE-Avoid freezing and thawing the Collagenase II sample. After withdrawing the Collagenase II sample, wipe off the outside of the pipette tip to remove any residual solution.]

3.1.1 Instrumental conditions

(See Ultraviolet-Visible Spectroscopy (857).)

Mode: UV

Analytical wavelength: 320 nm

Path length: 1 cm

Temperature: 25"

3.1.2 Analysis

Samples: Standard solution and Sample solutions

Transfer 1.0 mL of Substrate solution and 0.2 mL of Calcium chloride solution into a test tube, and equilibrate the test tube to 25". Start the reaction by adding 0.05 mL of Standard solution or each Sample solution. Prepare a blank by replacing the Standard solution or Sample solution with 0.05 mL of Tris buffer. Mix and incubate for exactly 15 min at 25". Transfer 0.5 mL of the reaction to the test tube containing 6.0 mL of Extraction mixture. Vortex immediately for 20 s. Transfer 3 mL of the ethyl acetate phase (upper layer) into a Drying tube using a glass pipette, and vortex thoroughly. Transfer the supernatant to a disposable, semi-micro cuvette suitable for UV absorbance with a Pasteur pipette. Record the absorbance.

Calculate the activity of collagenase II in units per milliliter:

Result = (A-A_B) x [V_TxV_E/(εxVxV_RxBxT)] x D

A = absorbance of the Standard solution or Sample solution

A_B = absorbance of the blank B

V_T = volume of the reaction mixture, 1.25 mL T

V_E = volume of ethyl acetate in the Extraction mixture, 5.0 mL E

ε = extinction coefficient for 320 nm, 21 (1 cm² µmol-1)

V = volume of the Standard solution or Sample solution, 0.05 mL

V_R = volume of the reaction transferred to Extraction mixture, 0.5 mL

B = absorption path length, 1 cm

T = incubation time, 15 min

D = dilution factor

[NOTE-One unit will release the equivalent of 1 µmol of PZ-Pro-Leu from PZ-Pro-Leu-Gly-Pro-D-Arg per minute under the conditions of the Assay.]

Calculate the specific activity of collagenase II in units per milligram of protein:

Result = Activity/C

Activity activity of collagenase II (units/mL)

C = protein concentration (mg/mL)

3.1.3 System suitability

Samples: Standard solution and Sample solutions

3.1.4 Suitability requirements

Average calculated activity: 85%-115% of the value on the label, Standard solution

Absorbance: 0.3-0.9, Standard solution and Sample solutions

Acceptance criteria: 10-16 units/mg of protein

4 PURITY

4.1 PROCEDURE

Solution A: 20 mM Tris and 1 mM calcium chloride pH 7.5, prepared as follows. Dissolve 2.42 g of Tris and 147 mg of calcium chloride dihydrate in 900 mL of water. Adjust with 2 N hydrochloric acid to a pH of 7.5. Dilute with water to a final volume of 1000 mL.

Solution B: 20 mM Tris, 1 mM calcium chloride, and 1 M sodium chloride pH 7.5, prepared as follows. Dissolve 2.42 g of Tris, 147 mg of calcium chloride dihydrate, and 58.44 g of sodium chloride in 900 mL of water. Adjust with 2 N hydrochloric acid to a pH of 7.5. Dilute with water to a final volume of 1000 mL.

Mobile phase: See Table 1.

Table 1

Storage buffer: 5 mM HEPES and 1 mM calcium chloride pH 7.5, prepared as follows. Dissolve 1.19 g of HEPES and 147 mg of calcium chloride dihydrate in 900 mL of water. Adjust with 4 N sodium hydroxide solution to a pH of 7.5. Dilute with water to a final volume of 1000 mL.

Standard solution: Thaw USP Collagenase II RS at room temperature shortly before use and mix. Store on ice or at 5". Dilute with Storage buffer to achieve a protein concentration of 5.5 mg/mL. Transfer to an HPLC vial and keep at 5". Prepare in duplicate, and inject each duplicate once.

Sample solution: Dilute Collagenase II with Storage buffer to achieve a protein concentration of 5.5 mg/mL and keep at 5".

Blank: Storage buffer

4.1.1 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 280 nm

Column: 5-mm x 5-cm; 10-µm packing 191

4.1.2 Temperatures

Autosampler: 5°

Column: 25°

4.2 Flow rate: 1.5 mL/min

Injection volume: 20 µL

4.2.1 System suitability

Sample: Standard solution

Suitability requirement: The chromatogram from the Standard solution corresponds to the typical chromatogram provided with USP Collagenase II RS.

4.2.2 Analysis

Sample: Sample solution

The Blank should be considered for integration. Evaluate the purity of Collagenase II using the percentage peak area method but excluding peaks associated with the Blank. All shoulders in the fronting and tailing of the main peak are integrated by dropping a perpendicular line at the inflection points and considered as separate impurities. Disregard any peaks having retention times greater than 25 min.

Acceptance criteria: NLT 90% for the main peak of collagenase II

5 IMPURITIES

CLOSTRIPAIN ACTIVITY

Potassium phosphate buffer: 0.1 M pH 7.6, prepared as follows. Dissolve 1.36 g of monobasic potassium phosphate in water, and dilute to 100 mL. Dissolve 2.28 g of dibasic potassium phosphate trihydrate in water, and dilute to 100 mL. Adjust the pH of the second solution to 7.6 with the first solution.

Dithiothreitol solution: 0.194 M, prepared as follows. Dissolve 60 mg of dithiothreitol (DTT) in 2 mL of Potassium phosphate buffer.

Calcium chloride solution: 0.01 M, prepared as follows. Dissolve 147 mg of calcium chloride dihydrate in 100 mL of water.

Substrate stock solution: 38 mM, prepared as follows. Dissolve 13 mg of N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE HCI) in 1 mL of Potassium phosphate buffer.

Substrate solution: 0.73 mM BAEE HCI, 7.8 mM DTT, and 0.4 mM calcium chloride, prepared as follows. Transfer 0.5 mL of Substrate stock solution, 1.0 mL of Dithiothreitol solution, and 1.0 mL of Calcium chloride solution to a 25-mL volumetric flask, and dilute with Potassium phosphate buffer to volume.

Sample solution: Prepare in such a way that AA/min lies in the 0.02-0.06 range. Dilute with ice-cold Potassium phosphate buffer if necessary.

4.2.3 Instrumental conditions

(See Ultraviolet-Visible Spectroscopy (857).)

Mode: UV

Analytical wavelength: 255 nm

Path length: 1 cm

Temperature: 25"

4.2.4 Analysis

Sample: Sample solution

Transfer 3.0 mL of Substrate solution into a cuvette, and equilibrate the cuvette to 25°. Start the reaction by adding 0.05 mL of Sample solution. Prepare a blank by replacing the Sample solution with 0.05 mL of Potassium phosphate buffer. Mix well. Determine the change in absorbance (AA/min) from the linear range of the reaction. Assay the

Sample solution in triplicate.

4.2.5 System suitability

Sample: Sample solution

Suitability requirement: 0.02-0.06 for AA/min

Calculate the activity of clostripain in units per milliliter in the portion of Collagenase II taken:

Result = [V_T/(ε x V_U x B)] x ΔA/ min × D

V_T = volume of the reaction mixture, 3.05 mL

ε = extinction coefficient for 255 nm, 0.81 (1 cm²-mmol¹)

V_U  = volume of the Sample solution, 0.05 mL

B = absorption path length, 1 cm

ΔA/ min × D = change in absorbance from the linear range of the reaction

D = dilution factor

Calculate the specific activity in units per milligram of protein:

Result = Activity/C

Activity =  activity of clostripain (units/mL)\

C = protein concentration (mg/mL)

Acceptance criteria: NMT 0.5 units of clostripain activity per milligram of protein

5.1 TRYPSIN ACTIVITY

Buffer: 0.1 M Tris and 0.02 M calcium chloride pH 8.0, prepared as follows. Dissolve 6.05 g of Tris and 1.45 g of calcium chloride dihydrate in 400 mL of water. Adjust with 2 N hydrochloric acid to a pH of 8.0 (at 25 ± 1°). Dilute with water to a final volume of 500 mL.

Substrate stock solution: Dissolve 10 mg of carbobenzoxy-valyl-glycyl-arginine-4-nitril-anilide acetate, accurately weighed, in 1.5 mL of water. Store on ice. [NOTE-Use freshly prepared solution only.]

Substrate solution: Prepare a solution by mixing 9.2 mL of Buffer and 1.0 mL of Substrate stock solution. Store on ice. [NOTE-Use freshly prepared solution only.]

Sample solution: Undiluted Collagenase II solution

5.1.1 Instrumental conditions

(See Ultraviolet-Visible Spectroscopy (857).)

Mode: Vis

Analytical wavelength: 405 nm

Path length: 1 cm

Temperature: 25°

5.1.2 Analysis

Sample: Sample solution

Transfer 1.02 mL of Substrate solution into a polystyrene semi-micro cuvette, allow the temperature to stabilize, equilibrate the cuvette to 25", and wait for 10 min. Start the reaction by adding 0.10 mL of Sample solution. Start recording the absorbance and continue for at least 5 min after the addition of the Sample solution. Determine the change in absorbance (AA/min) from the linear range of the reaction. Assay the Sample solution in triplicate. [NOTE-Use polyethylene pipette tips to transfer the Sample solution. The pipette tip should not be wet before transfer, and each pipette tip should be used only for transferring one sample. After withdrawing the Sample solution, wipe off the outside of the tip to remove any residual solution. After adding the Sample solution to the Substrate solution, rinse the tip by pipetting the solution up and down 2-3 times, discard the tip, and mix.]

5.1.3 System suitability

Sample: Sample solution

Suitability requirement: >0.01 for AA/min

Calculate the activity of trypsin in units per milliliter in the portion of Collagenase II taken:

Result = [V_T/(ε x V_U x B)] x AA/min x D

V_T = volume of the reaction mixture, 1.12 mL T

ε = extinction coefficient for 405 nm, 10.4 (1 cm² mmol¹)

V_U = volume of the Sample solution, 0.10 mL

B = absorption path length, 1 cm

ΔA/ min = change in absorbance from the linear range of the reaction

D = dilution factor

Calculate the specific activity in units per milligram of protein:

Result = Activity/C

Activity = activity of trypsin (units/mL)

C = protein concentration (mg/mL)

Acceptance criteria: NMT 0.5 units of trypsin activity per milligram of protein

6 SPECIFIC TESTS

6.1 PROTEIN CONTENT

Sample solutions: Dilute Collagenase II in water. Prepare at least in triplicate. (NOTE-Prepare the dilution using plastic pipette tips and not glass pipettes. Carefully wipe off the outside of the tip to remove any residual solution.]

Blank solution: Water

Instrumental conditions (See Ultraviolet-Visible Spectroscopy (857).)Mode: UV

Analytical wavelength: 280 nm

Path length: 1 cm

6.1.1 System suitability

Samples: Sample solutions

Suitability requirement: Absorbance is in the range of 0.10-1.00.

6.1.2 Analysis

Samples: Sample solutions and Blank solution Determine the net absorbance of Sample solutions by subtracting the absorbance of the Blank solution from the absorbance of each Sample solution. Determine the average net absorbance of Sample solutions.

Calculate the protein concentration in mg/mL:

Result = A_U × D/ε

A_U = average net absorbance of the Sample solutions

D = dilution factor

ε = extinction coefficient for 280 nm, 1.4 (mLmg1cm-1)

BACTERIAL ENDOTOXINS TEST (85): NMT 50 USP Endotoxin Units/mg of protein

Change to read:

MICROBIAL ENUMERATION TESTS (61): The total (IRA 1-Sep-2022) count is NMT 100 cfu/mL.

7 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Store in closed containers at -60° to-90".

LABELING: The labeling states that the material is derived from Clostridium histolyticum along with the lot number, product or catalog number, and storage conditions.

Change to read:

USP REFERENCE STANDARDS (11)

USP Collagenase II RS

(IRA 1-Sep-2022)